Microbial Symbionts Accelerate Wound Healing via the Neuropeptide Hormone Oxytocin

Wound healing capability is inextricably linked with diverse aspects of physical fitness ranging from recovery after minor injuries and surgery to diabetes and some types of cancer. Impact of the microbiome upon the mammalian wound healing process is poorly understood. We discover that supplementing the gut microbiome with lactic acid microbes in drinking water accelerates the wound-healing process to occur in half the time required for matched control animals. Further, we find that Lactobacillus reuteri enhances wound-healing properties through up-regulation of the neuropeptide hormone oxytocin, a factor integral in social bonding and reproduction, by a vagus nerve-mediated pathway. Bacteria-triggered oxytocin serves to activate host CD4+Foxp3+CD25+ immune T regulatory cells conveying transplantable wound healing capacity to naive Rag2-deficient animals. This study determined oxytocin to be a novel component of a multi-directional gut microbe-brain-immune axis, with wound-healing capability as a previously unrecognized output of this axis. We also provide experimental evidence to support long-standing medical traditions associating diet, social practices, and the immune system with efficient recovery after injury, sustained good health, and longevity.     

Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

Release of poly a(+) messenger RNA from rat liver rough microsomes upon disassembly of bound polysomes

Several procedures were used to disassemble rat liver rough microsomes (RM) into ribosomal subunits, mRNA, and ribosome-stripped membrane vesicles in order to examine the nature of the association between the mRNA of bound polysomes and the microsomal membranes. The fate of the mRNA molecules after ribosome release was determined by measuring the amount of pulse-labeled microsomal RNA in each fraction which was retained by oligo-dT cellulose or by measuring the poly A content by hybridization to radioactive poly U. It was found that ribosomal subunits and mRNA were simultaneously released from the microsomal membranes when the ribosomes were detached by: (a) treatment with puromycin in a high salt medium containing Mg++, (b) resuspension in a high salt medium lacking Mg++, and (c) chelation of Mg++ by EDTA or pyrophosphate. Poly A-containing mRNA fragments were extensively released from RM subjected to a mild treatment with pancreatic RNase in a medium of low ionic strength. This indicates that the 3' end of the mRNA is exposed on the outer microsomal surface and is not directly bound to the membranes. Poly A segments of bound mRNA were also accessible to [(3)H] poly U for in situ hybridization in glutaraldehyde-fixed RM. Rats were treated with drugs which inhibit translation after formation of the first peptide bonds or interfere with the initiation of protein synthesis. After these treatments inactive monomeric ribosomes, as well as ribosomes bearing mRNA, remained associated with their binding sites in microsomes prepared in media of low ionic strength. However, because there were no linkages provided by nascent chains, ribosomes, and mRNA, molecules were released from the microsomal membranes without the need of puromycin, by treatment with a high salt buffer containing Mg++. Thus, both in vivo and in vitro observations are consistent with a model in which mRNA does not contribute significantly to the maintenance of the interaction between bound polysomes and endoplasmic reticulum membranes in rat liver hepatocytes.     

The identification of dimethylphenylarsine as a microbial metabolite using a simple method of chemofocusing

Candida humicola acts on benzenearsonic acid to produce dimethylphenylarsine, which was identified by mass spectroscopy following the chemofocusing of the volatile metabolite onto a mercuric chloride impregnated filter. The same technique established that trimethylarsine is the volatile metabolic product obtained from C. humicola treated with 4-NH₂-2-OHC₆H₃AsO(OH)₂ and (CH₃)₃AsO. Arsanilic acid, 4-NH₂C₆H₄AsO(OH)₂, is not metabolized to a volatile arsine.     

6R instrumented spatial linkages for anatomical joint motion measurement--Part 2: Calibration.

The six-revolute-joint instrumented spatial linkage (6R ISL) is often the measurement system of choice for monitoring motion of anatomical joints. However, due to tolerances of the linkage parameters, the system may not be as accurate as desired. A calibration algorithm and associated calibration device have been developed to refine the initial measurements of the ISL's mechanical and electrical parameters so that the measurement of six-degree-of-freedom motion will be most accurate within the workspace of the anatomical joint. The algorithm adjusts the magnitudes of selected linkage parameters to reduce the squared differences between the six known and calculated anatomical position parameters at all the calibration positions. Weighting is permitted so as to obtain a linkage parameter set that is specialized for measuring certain anatomical position parameters. Output of the algorithm includes estimates of the measuring system accuracy. For a particular knee-motion-measuring ISL and calibration device, several interdependent design parameter relationships have been identified. These interdependent relationships are due to the configuration of the ISL and calibration device, the number of calibration positions, and the limited resolution of the devices that monitor the position of the linkage joints. It is shown that if interdependence is not eliminated, then the resulting ISL parameter set will not be accurate in measuring motion outside of the calibration positions, even though these positions are within the ISL workspace.     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

Association of variable <Emphasis Type="Italic">rs1801282</Emphasis> locus of <Emphasis Type="Italic">PPARG2</Emphasis> gene with diabetic nephropathy

The association of the variable rs1801282 locus of the PPARG2 gene (peroxisome proliferator-activated receptor gamma) with type 2 diabetes mellitus and its complications was analyzed in inhabitants of the Republic of Bashkortostan. The genotype frequencies of the variable rs1801282 locus of the PPARG2 gene did not significantly differ in groups of healthy persons and patients with type 2 diabetes in all three considered inheritance models (codominant, dominant, and recessive). At the same time, it was demonstrated that the risk of one of the diabetic complications, i.e., diabetic nephropathy, was associated with the variable rs1801282 locus of the PPARG2 gene. Diabetic nephropathy was more common in patients with the C/C genotype (62.7%) compared to the C/G and G/G genotypes (37.5%), P = 0.036. The G allele is protective in regard to diabetic nephropathy (OR = 0.36) in patients with type 2 diabetes mellitus.     

Different Rab GTPases associate preferentially with alpha or beta GDP-dissociation inhibitors

GDIs (GDP-dissociation inhibitors) bind to Rab GTPases and mediate their membrane targeting and recycling. In vitro, most Rabs can bind to either of the major isoforms of GDI, leading to the assumption that the proportion of each specific Rab/GDI complex in vivo reflects the relative abundance of the alpha versus beta forms of GDI. Here we show that when human teratocarcinoma cells (Ntera2) are induced to differentiate into postmitotic neurons (NT2N), there is a major change in the proportion of GDIalpha relative to GDIbeta. Under these conditions, certain Rab GTPases associate preferentially with either GDIalpha or GDIbeta, irrespective of the relative abundance of the GDI isoform. These findings suggest that heretofore unrecognized functional specificity may exist between the two major forms of GDI.     

Design of an MRI-compatible robotic stereotactic device for minimally invasive interventions in the breast

The objective of this work was to develop a robotic device to perform biopsy and therapeutic interventions in the breast with real-time magnetic resonance imaging (MRI) guidance. The device was designed to allow for (i) stabilization of the breast by compression, (ii) definition of the interventional probe trajectory by setting the height and pitch of a probe insertion apparatus, and (iii) positioning of an interventional probe by setting the depth of insertion. The apparatus is fitted with five computer-controlled degrees of freedom for delivering an interventional procedure. The entire device is constructed of MR compatible materials, i.e. nonmagnetic and non-conductive, to eliminate artifacts and distortion of the MR images. The apparatus is remotely controlled by means of ultrasonic motors and a graphical user interface, providing real-time MR-guided planning and monitoring of the operation. Joint motion measurements found probe placement in less than 50 s and sub-millimeter repeatability of the probe tip for same-direction point-to-point movements. However, backlash in the rotation joint may incur probe tip positional errors of up to 5 mm at a distance of 40 mm from the rotation axis, which may occur for women with large breasts. The imprecision caused by this backlash becomes negligible as the probe tip nears the rotation axis. Real-time MR-guidance will allow the physician to correct this error Compatibility of the device within the MR environment was successfully tested on a 4 Tesla MR human scanner